Analysis of a cell-cycle promoter bound by a response regulator

Caulobacter crescentus CtrA protein, an OmpR-type response regulator, receives cell-cycle signals and binds the proposed consensus TTAA-N7-TTAA present inside the chromosome replication origin and cell-cycle transcription promoters. We synthesized a 42 bp cell-cycle promoter based on this consensus and elements of the fliL promoter. Over 100 promoter mutations were assayed for transcription directed by CtrA. Although both CtrA binding half-sites cooperate and the N7 spacing is critical for transcription, the upstream half-site is relatively flexible. The downstream CtrA half-site is less flexible and more important for cell-cycle regulation. A CtrA binding site and a -10 promoter element are sufficient for cell-cycle transcription, and both sequences cooperate and compensate for respective defects. Mutations in the CtrA binding site, but not in the -10 promoter sequence, perturb cell-cycle transcription. A single base-pair change switches a cell-cycle promoter into a strong conventional promoter. We propose rules for CtrA binding and promoter interactions implying how CtrA evolved into a versatile regulator of cell-cycle functions including flagellar biogenesis, cell division, DNA methylation, and chromosome replication.